2.1. Cell Culture and VSMC-Derived Foam Cell Formation
Human aortic vascular smooth muscle cells (VSMCs, #6110) and smooth muscle cell medium (SMCM, 1101) were purchased from ScienCell (San Diego, CA, USA). VSMCs were cultured in SMCM containing 2% fetal bovine serum, 1% smooth muscle cell growth supplement (SMCGS), 100 units/mL penicillin, and 100 µg/mL streptomycin, at 37°C in a 5% CO2 humidified atmosphere. VSMCs between passages three and six were used in all experiments. After reaching 70% confluence, VSMCs were synchronized in fetal bovine serum- and SMCGS-free SMCM and cultured at 37°C in a 5% CO2 humidified atmosphere for 24 h. Cholesterol–methyl-β-cyclodextrin (C4951; Sigma, Saint Louis, MO, USA) was dissolved in SMCGS-free SMCM to a concentration of 5 μg/mL; the cell medium was replaced with this solution, and the cells were cultured for 72 h.